CN103620376B - A method for observing a sample of - Google Patents

A method for observing a sample of Download PDF

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CN103620376B
CN103620376B CN 201280019367 CN201280019367A CN103620376B CN 103620376 B CN103620376 B CN 103620376B CN 201280019367 CN201280019367 CN 201280019367 CN 201280019367 A CN201280019367 A CN 201280019367A CN 103620376 B CN103620376 B CN 103620376B
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surface
light
object
sample
droplets
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CN 201280019367
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CN103620376A (en )
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马蒂厄·杜波伊
皮埃尔·乔利
弗莱德瑞克·马勒拉瑞德
皮埃尔·马库斯
拉斐尔·马泰
阿尔梅·诺维利-卢梭
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原子能与替代能源委员会
生物梅里埃公司
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6486Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03Cuvette constructions
    • G01N2021/0346Capillary cells; Microcells
    • G01N2021/035Supports for sample drops
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65Raman scattering

Abstract

本发明涉及一种用于观察样品的方法,所述方法包括以下步骤:a)提供基本上透明的介质形成的物体(G,V),所述介质具有第一折射率(nint)且包括凸形、圆形或多平面的第一表面(S1)和基本上平坦的第二表面(S2),所述第一表面将所述物体与周围介质(MA)分离开,所述周围介质具有低于所述第一折射率的第二折射率(next),待观察的所述样品包括在所述物体内或沉积在所述第一表面上;b)通过所述基本上平坦的第二表面观察所述样品;以及c)通过在所述步骤b)期间检测与所述物体相对应的光环,确定所述样品中的物质或对象的存在。 The present invention relates to a method for observing a sample, said method comprising the steps of: a) providing a substantially transparent article (G, V) formed in the medium, said medium having a first refractive index (NINT) and comprises a convex shaped, round or planar first surface (S1) and a substantially planar second surface (S2), the first surface of the object and the surrounding medium (MA) separated from the ambient medium has a low said second refractive index of the first (next), the sample to be observed or the object included in deposited on the first surface; b) through said second substantially planar surface viewing the sample; and c) detecting said object during said step b) the corresponding halo, determining the presence of a substance in a sample or subject.

Description

用于观察样品的方法 A method for observing a sample of

技术领域 FIELD

[0001] 本发明涉及一种用于观察样品的方法,尤其用于生物或化学应用,例如,用于检测微生物或对微生物计数以及研究化学反应的动力学。 [0001] The present invention relates to a method for observing a sample, in particular for biological or chemical applications, e.g., for the detection of microbes or microbial count and kinetic studies of chemical reactions.

背景技术 Background technique

[0002] 根据优选实施方式的方法基于使用包含待观察样品的微滴或所述样品本身。 [0002] The method itself is based on a preferred embodiment of droplets containing the sample or the sample to be observed. 由AB Theberge 等所著的文章“Microdroplets in Microfluidics : An Evolving Platform for Discoveries in Chemistry and Biology”(Angew Chem Int Ed20I0,49,5846-5868) 综述了用于化学和生物学中的各种应用中的基于使用液态微滴的微流体技术。 The article written by the AB Theberge, etc. "Microdroplets in Microfluidics: An Evolving Platform for Discoveries in Chemistry and Biology" (Angew Chem Int Ed20I0,49,5846-5868) reviewed for various chemical and biological applications a liquid droplet-based microfluidics. 术语“微滴” 指直径(或通常为主要尺寸)的范围为Iym至ΙΟΟμπι的液滴。 The term "droplets" refers to the diameter (or major dimension is typically) in the range of Iym to ΙΟΟμπι droplets.

[0003] 文献US2005/0221339描述了一种用于对在微流体系统中操作的微滴中的化学成分进行光学检测的方法。 [0003] Document US2005 / 0221339 describes a method for the chemical composition of the droplet operations in a microfluidic system for optical detection.

[0004] 由B Rotman所著的文章“Measurement of activity of single molecules of β-D-Galactosidase”,美国国家科学院院刊(Proceedings of the Natl Acad Sci USA), 1961,47,第1981-1991页,描述了一种用于检测酶的分离的分子的方法。 [0004] "Measurement of activity of single molecules of β-D-Galactosidase", PNAS (Proceedings of the Natl Acad Sci USA), 1961,47, pages written by the B Rotman article of 1981-1991, It describes a method of separating molecules for detecting enzyme. 在该方法中,包含酶(高度稀释的)和用于该酶的荧光基质的水溶液被喷到在两个显微镜用载玻片之间限定的小体积的硅酮油中。 In the method, comprising the enzyme (highly diluted) and an aqueous solution of a fluorescent substrate for the enzyme is sprayed in a small volume of silicone oil between two microscope slide as defined in. 考虑到该硅酮油密度小于水且不与水混溶,该溶液形成置于下显微镜用载玻片的上表面上的微滴。 Considering that the silicone oil is less dense than water and not miscible with water, the solution forms droplets on the microscope slide is placed on the surface. 然后,借助显微镜,通过显微荧光测定对液滴进行分析。 Then, by means of a microscope and analyzed by measuring the fluorescence of the microscopic droplets. 包括该酶的至少一个分子的液滴显现为发光盘,而不包含所述分子的液滴显现为发光环的形式。 Including droplets of at least one molecule of the enzyme appears to be made of an optical disc, comprising the molecule does not appear as light ring droplet form.

[0005] 这种方法的主要限制在于由荧光发出的光的强度低。 [0005] The main limitation of this approach is the low intensity of light emitted by the phosphor. 当液滴中包含的微观尺寸的对象对光的散射(弹性散射或拉曼散射)被观察到时,出现类似的问题。 When the droplet size of the object contained in the microscopic light scattering (elastic scattering or Raman scattering) is observed, similar problems.

发明内容 SUMMARY

[0006] 本发明的目的是通过提出一种用于观察样品的配置克服这种限制,这种配置提高了由样品发出或散射的光的收集效率。 [0006] The object of the present invention is to provide a configuration by observing the sample for overcoming such limitations, this configuration improves the collection efficiency of the light emitted or scattered by the sample. 然而,本发明不限于使用液滴,而包括液滴由固体介质或凝胶形成的物体代替的情况。 However, the present invention is not limited to the droplet, the droplet comprising a case body formed by a solid or gel medium replaced.

[0007] 因此,在一方面,本发明提供了一种用于观察样品的方法,所述方法包括以下步骤: [0007] Accordingly, in one aspect, the present invention provides a method for observing a sample, said method comprising the steps of:

[0008] a)提供基本上透明的介质的物体,所述基本上透明的介质具有第一折射率叫的且具有凸形、圆形或多平面的第一表面和基本上平坦的第二表面,所述第一表面将所述物体与周围介质分离开,所述周围介质具有第二折射率rwt,待观察的所述样品由所述物体构成或位于所述物体内或沉积在所述第一表面上;以及 [0008] a) providing a substantially transparent object medium, the substantially transparent medium having a second surface and having a first index of refraction called convex, round or planar first surface and a substantially planar , separated from the first surface of the object and the surrounding medium, the refractive index of the surrounding medium has a second RWT, the sample to be observed or constituted by the object located within the object deposited on said first or an upper surface;

[0009] b)观察所述样品; [0009] b) viewing the sample;

[0010] 其特征在于: [0010] wherein:

[0011] -在步骤b)期间,通过所述基本上平坦的第二表面观察所述样品;以及 [0011] - during steps b), observed by a substantially planar second surface of the said sample; and

[0012] -所述第二折射率低于所述第一折射率。 [0012] - the second refractive index lower than the first index of refraction.

[0013] 所述方法可以包括步骤c),所述步骤c)用于根据在步骤b)期间观察所述样品的结果来确定所述样品中的物质或对象的存在。 [0013] The method may include the step c), said step c) for determining the presence of a substance in a sample or object in accordance with result of the observation of the sample during step b). 尤其是,对象可以是生物物种,如微生物,例如, 细菌、酵母、真菌、病毒等。 In particular, the object may be a biological species, such as microorganisms, e.g., bacteria, yeast, fungi, viruses and the like. 例如,待检测的物质可以溶解或分散在透明或半透明的介质中。 For example, the substance to be detected may be dissolved or dispersed in a transparent or translucent medium.

[0014] 样品可以是液态的,例如,生物液体、离子性液体、水溶液或凝胶。 [0014] The sample may be liquid, e.g., biological fluid, an ionic liquid, an aqueous solution or a gel. 在凝胶的情况下,例如,粘度可以在IcP (厘泊)至IOOOcP范围内,S卩,在HT3Pa · s至IPa · s范围内。 In the case of a gel, for example, the viscosity may ICP (centipoise) into the range IOOOcP, S Jie, in HT3Pa · s to IPa · s range.

[0015] 将在下面更详细地描述,周围介质的折射率低于所述物体的折射率确保沿着凸形的所述第一表面(“光碗”)部分地导引由所述样品发射或散射的光,这提高了在平坦的第二表面这一侧上的光的检测效率。 [0015] will be described in more detail below, a refractive index lower than the refractive index of the medium surrounding the object to ensure that the guide part along the sample emitted by said first convex surface ( "light bowl") or scattered light, which improves the detection efficiency of the light on the second side of the planar surface. 与此相反,在现有技术(尤其参见上面引用的B Rotman的文章)中,通常使用具有较高的光学折射率的油中的水溶液的“反相乳液”,和/或通过凸面观察样品。 In contrast, in the prior art (see, in particular B Rotman above cited articles) typically used oil has a higher optical index of refraction of the "inverse emulsion" aqueous solution and / or sample observation by a convex surface.

[0016] 通常,该物体的直径或主要尺寸可以在ΙμΐΉ至Imm范围内,优选在ΙΟμπι至500μηι的范围内。 [0016] Typically, the diameter or major dimension of the object may be in the ΙμΐΉ to Imm, preferably in the range of ΙΟμπι to 500μηι.

[0017] 根据本发明的各种实施方式: [0017] According to various embodiments of the present invention:

[0018] -所述物体可以是基本上半球形的形状。 [0018] - The object may be a substantially hemispherical shape. 这实际是使由样品发射或散射的光的收集最大化的几何形状; This is actually light scattered or emitted by the sample to maximize the collection of geometry;

[0019] -所述物体可以是液滴,所述液滴附着到透明基板上而形成所述基本上平坦的第二表面; [0019] - The object may be a liquid droplet, the droplets adhere to a transparent substrate to form a second surface of the substantially planar;

[0020] -所述步骤a)可以包括产生形成多个所述物体的多个液滴,所述多个液滴中的至少一些液滴包括至少一个待检测的对象或物质;以及所述步骤b)可包括同时观察多个所述液滴; [0020] - the step a) may include generating a plurality of droplets of said plurality of objects, at least some of the droplets of the plurality of droplets comprise at least one object or substance to be detected; and said step b) may comprise a plurality of said droplets were observed;

[0021] -与此相反,所述物体可以是固体,在这种情况下,所述样品可以由沉积在所述物体的第一表面上的层构成。 [0021] - In contrast to this, the object may be solid, in which case, the sample may be constituted by a first layer deposited on the surface of the object. 有利地,所述层可以是对分析物敏感的官能化层,所述分析物可能包括在所述周围介质中。 Advantageously, the functional layer may be a layer sensitive to the analyte, the analyte may be included in the surrounding medium. 当所述物体是固体时,所述样品还可以是放置成与所述物体的第一表面接触的液态或粘性的样品; When the object is a solid, the sample may also be placed in a liquid or viscous sample in contact with a first surface of the object;

[0022] -所述物体可以由对分析物敏感的材料形成,所述分析物可能包括在所述周围介质中; [0022] - The object may be formed from a material sensitive to the analyte, the analyte may be included in the surrounding medium;

[0023] -所述周围介质可以是空气; [0023] - The medium may be ambient air;

[0024] -所述步骤b)可以包括照射所述物体的操作。 [0024] - step b) may include an operation of the object is irradiated. 优选地,所述照射可以通过所述基本上平坦的第二表面进行,以使其进而可以从上述的光导引效应受益。 Preferably, the irradiation can be carried out by a second surface of said substantially planar, so that it in turn can benefit from the effect of light guiding. 在不同的变型中: In a different variant:

[0025] -所述步骤b)可以包括检测光信号,例如,荧光信号。 [0025] - the step b) may comprise detecting an optical signal, e.g., a fluorescent signal. 在这种情况下,所述步骤b)可以通过落射荧光显微术进行; In this case, step b) can be carried out by epifluorescence microscopy;

[0026] -所述步骤b)可以包括检测拉曼散射信号; [0026] - the step b) may comprise detecting a Raman scattering signal;

[0027] -所述步骤b)可以通过反射显微术或透射显微术进行。 [0027] - step b) can be carried out by reflection microscopy or transmission microscopy.

附图说明 BRIEF DESCRIPTION

[0028] 根据结合附图进行的示例性描述,本发明的其它特征、细节和优点将变得清楚,这些附图分别表示: [0028] The exemplary description in conjunction with the accompanying drawings, other features of the present invention, details and advantages will become apparent, these figures represent:

[0029] -图1示出在液滴和具有较低折射率的周围介质(如空气)之间的界面处传播的各种光线路径; [0029] - Figure 1 shows a variety of light propagation at the interface between the droplets and the surrounding medium (e.g., air) having a lower refractive index;

[0030] -图2A和图2B示出从液滴的位于所述界面附近的位置获得的光线样式; [0030] - Figures 2A and 2B shows a pattern obtained from the light located near the position of the interface of the droplets;

[0031] -图3是示出在穿过光源且垂直于所述第二表面的平面中,在该液滴的边缘附近进入该液滴的光的范围的光线样式; [0031] - Figure 3 is a diagram illustrating the light source and passing through a plane perpendicular to said second surface, the light entering the liquid droplets in the vicinity of the edge of the range of droplet light pattern;

[0032] -图4A和图4B示出上文所引用的由B Rotman所著文章中描述的观察设置的两个光线样式; [0032] - Figures 4A and 4B shows two light patterns observed are described by B Rotman written article cited above provided;

[0033] -图5是示出本发明的优点的对比试验; [0033] - Figure 5 is a diagram illustrating comparative tests the advantages of the present invention;

[0034] -图6示出用于实施根据本发明的方法的实验装置; [0034] - Figure 6 shows the experimental apparatus for implementing the method according to the invention;

[0035] -图7A和图7B分别示出根据本发明的第一实施方式的方法的示意图和使用该方法获得的图像; [0035] - FIGS. 7A and 7B show an image obtained according to the method used and a schematic embodiment of the method of the first embodiment of the present invention;

[0036] -图8A和图8B分别示出根据本发明的第二实施方式的方法的示意图和使用该方法获得的图像; [0036] - Figures 8A and 8B illustrate the image obtained by the method according to a second embodiment illustrating a method embodiment of the present invention and use;

[0037] -图9A和图9B分别示出根据本发明的第三实施方式的方法的示意图、使用该方法获得的图像以及这样的图像的细节; [0037] - FIGS. 9A and 9B are a schematic view showing a third embodiment of a method embodiment of the present invention, the image and the details of such an image is obtained using this method;

[0038] -图10是用于根据本发明的第四实施方式的方法的示意图; [0038] - FIG. 10 is a schematic embodiment of the method according to the fourth embodiment of the present invention is used;

[0039] -图11是用于根据本发明的第五实施方式的方法的示意图;以及 [0039] - Figure 11 is a schematic diagram of the method according to the fifth embodiment of the present invention; and

[0040] -图12A至图12D示出物体形状相对于半球的偏差的结果。 [0040] - Figures 12A to 12D show the shape of the object with respect to skew the results of the hemisphere.

具体实施方式 detailed description

[0041] 图1示出本发明所基于的物理现象。 [0041] FIG. 1 shows the physical phenomena of the present invention is based. 考虑(实际)折射率为mnt的液滴G浸在(实际) 折射率为next〈mnt的周围介质MA中。 Consider (actual) a refractive index of the immersion mnt droplets G (actual) a refractive index of next <mnt MA in the surrounding medium. 在水滴在空气中的情况下,mnt=l .33〈next=l。 In the case of water droplet in air, mnt = l .33 <next = l.

[0042] 根据表面张力效应,液滴呈现为直径为R的基本上球形或截球形的形状,如果R为约几微米,由重力引起的变形在实际中可以忽略。 [0042] The surface tension effects, droplets exhibit substantially spherical or frusto-spherical shape of a diameter of R, if R is about several microns, the deformation caused by gravity can be ignored in practice.

[0043] 图1示出在液滴G内传播的三条光线Rl、R2和R3,且这三条光线分别以入射角Q1J2 和θ3触及液滴表面S1。 [0043] Figure 1 shows a three Rl light propagating in the droplets G, R2 and R3, respectively, and these three rays reach an incident angle θ3 and Q1J2 droplet surface S1. 通常,入射角被定义为光线与表面的法线之间所形成的角。 Typically, the angle of incidence is defined as the angle between a normal ray and the surface.

[0044] 所有以大于临界角Θ。 [0044] In all Θ is greater than the critical angle. (由 (by

Figure CN103620376BD00061

给出,)的入射角触及表面Sl的光线发生全反射,且保持留在液滴内部。 Given) touches the incident angle of the total reflection surface of the light Sl and the left inside the droplet held. 在图1的示例中,光线Rl (其中,Gi=D和光线R2(其中,θ2>θ。)就是这种情况,而由于θ3〈θ。,光线R3离开液滴。 In the example of Figure 1, the rays Rl (wherein, Gi = D and the light R2 (where, θ2> θ.) This is the case, and because θ3 <θ., Ray R3 leave droplets.

[0045] 简单的几何推理可以证明发生全反射的光线保持局限在液滴表面Sl附近,且距离液滴的中心的距离不能小于距离 [0045] The simple geometric reasoning may prove totally reflected light remains localized near the droplet surface Sl, and can not be smaller than the distance from the center of the droplet

Figure CN103620376BD00062

[0046] 为了理解这种效果如何能够有助于对对象(如微生物或荧光分子)进行观察,考虑在图2中所示的情况,其示出了附着到透明支持体SS且浸在介质MA (如,空气)中的半球状液滴G。 [0046] To understand how this effect can contribute to the object (e.g., a microorganism or a fluorescent molecule) observation, consider the case shown in FIG. 2, which shows the adhesion to the transparent support and immersed in SS medium MA (e.g., air) in a hemispherical droplet G. 和前面的情况一样,next〈runt,基板的折射率nss可以小于mnt或大于mnt;优选地,nss不会太高(例如,小于2mnt或等于2mnt),以使基板本身不封堵光线。 And the previous case, the refractive index nss next <runt, the substrate may be less than or greater than mnt mnt; Preferably, NSS is not too high (e.g., less than or equal to 2mnt 2mnt), so that the substrate itself does not block the light. 光线由位于靠近液滴的半球形表面Sl的位置处的荧光分子发出(S2表示与基板的接触面)。 Fluorescent molecules at the location of the light from the semi-spherical surface located adjacent droplets emitted Sl (S2 represents the contact surface of the substrate). 可以看出,这些光线相当大部分由空气-水界面导向成呈“光碗” BL的形状的层。 As can be seen, a substantial portion of these rays by the air - water interface to guide a laminar shape "light bowl" BL's. 这些光线在平坦表面S2的包括在内半径 These rays included planar surface S2 radius

Figure CN103620376BD00071

和外半径R之间的环形形状的区域(其也可被称作圆环)中离开液滴。 And the region of the annular shape between the outer radius R (which may also be referred to as a ring) in droplets to leave. 可以理解,这提高了光收集效率。 It is understood that this improves the light collection efficiency. 光碗BL (其也可由术语球形包络表示)的厚度等于Rr。 Bowl BL optical thickness (which is also represented by the term spherical envelope) is equal to Rr.

[0047] 为了确定导向成光碗的光束的角度范围,图2B涉及穿过位于与中心C距离为rP的位置处的固定点P的光线。 [0047] In order to determine the angular range of the guide light beam bowl, FIG. 2B is located at the center of the light passes through the fixed point C at a distance of P rP position relates. 如果rP满足条件r〈rP〈R,那么,可以示出,存在两条光线RLl和RL2, 光线RLl和光线RL2以入射临界角Θ。 RP if the condition r <rP <R, then it can be shown, there are two light RLl and RL2, RL2 light rays RLl and the critical angle of incidence Θ. 接触圆Sl,且光线RLl和光线RL2的标记为RP的平分线与CP正交。 Contact circles Sl, and the light rays RLl and RL2 labeled RP CP perpendicular to the bisector. 在RP和RLl或RL2之间的角度ΘΝΑ满足 The angle between the RP RLl and RL2 ΘΝΑ satisfied or

Figure CN103620376BD00072

在光学几何方面,这表明在物体G内部,由通过P的光线所形成的光束具有主光线RP(垂直于CP)和孔径角ΘΝα』να的极限值为:0(当rp=r时)和 In terms of optical geometry, which indicates that the interior of the object G, the light beam formed by light having a principal ray P RP (perpendicular to the CP) and aperture angle ΘΝα "να limit value is: 0 (when rp = r), and

Figure CN103620376BD00073

(当rp=R时)。 (When rp = R). 因此,光束在环边缘具有最大数值孔径。 Thus, the light beam having the maximum numerical aperture at the edge of the ring.

[0048] 图3示出相反情况,其中,光源SL通过基板SS照射液滴G。 [0048] FIG. 3 illustrates the opposite situation, wherein the light source SL is irradiated through the substrate SS droplets G. 优选地,在光源SL和透明基板SS之间没有遮罩的情况下照射。 Preferably, the irradiation without mask between the light source and the transparent substrate of SS SL. 此外,在“暗场”设置下基板SS和液滴G未被照射。 Further, in the "dark field" is set SS and the substrate G is not irradiated droplets. 在该图中,从平坦表面S2经由上述环形区域穿入液滴的光线保持留在“光碗”BL中。 In this figure, the penetration of droplets from a flat annular surface S2 via the left in the holding area light "optical bowl" BL in. 与不发生光封堵相比,位于该“碗”内部的样品或样品的一部分被更有效地照射。 Compared with the light blocking does not occur, which is located "bowl" of the interior of the sample or sample portion is irradiated more efficiently.

[0049] 限制光线的该效果取决于条件next〈mnt,这可在图4A和图4B中看到,图4A和图4B涉及其中n' ext>n' int这一相反情况(水滴G'(mnt'〜1.33)在硅酮油浴MA'(next'〜1.44)中的情况)。 [0049] This effect limits the light depends on the conditions next <mnt, which can be seen in FIGS. 4A and 4B, FIGS. 4A and 4B relate wherein n 'ext> n' int the contrary (droplets G '( mnt'~1.33) in the case of a silicone oil bath MA '(next'~1.44) in). 在这些图中可以看出,在这种情况下,无论是由外部源SL发射的光线还是从液滴表面上的位置所获得的光线都不被封堵。 As can be seen in these figures, in this case, either the light emitted by an external light source SL is obtained from the position of the droplet on the surface it is not blocked.

[0050] 图5可用于将本发明的观察配置(Cl)与两个其他配置进行比较,这两个配置为:液滴被挤压在下显微镜用载玻片和上盖玻片之间(C2),和液滴置于载玻片上并从液滴的凸面侧观察液滴(C3)。 [0050] FIG. 5 may be used to observe the arrangement of the present invention (Cl) is compared with two other configurations, both configured to: lower the droplets are pressed between microscope slides and coverslips (C2 ), and placed on a slide and observed droplet droplets (C3) from the convex side of the droplet. 在所有情况下,液滴由5μΜ的荧光团4MU (4-甲基伞形酮)的水溶液构成。 In all cases, the droplets is constituted by an aqueous solution of (4-methylumbelliferone) fluorophores 5μΜ of 4MU. 通过落射荧光显微术(即,利用激发和从顶部检测)来观察液滴。 Droplets observed by epifluorescence microscopy (i.e., with excitation and detection from the top). 可以看出,本发明的配置Cl可用于观察到非常明亮的环,而在其它配置中,荧光液滴呈现为在背景下很难看到的亮度非常低的盘。 As can be seen, the configuration of the present invention may be used Cl observed very bright ring, while in other configurations, the phosphor brightness of droplets presented in the context of the hard disk to see very low. 实验测量表明,与Rotman使用的“水在油中”配置相比,本发明的配置可以使收集的光量提高20倍。 Experimental measurements indicated that the "water in oil" as compared to the use configuration and Rotman, the present invention may be arranged so that the amount of light collected 20 times. 术语“光量”是指在整个滴(包括明亮环和暗得多的中心区域)上测量的光量。 The term "light amount" refers to the amount of light in the entire drop (including the bright ring and the dark central area much) on the measurement. 相比之下,如果仅考虑半径在 In contrast, if only consider radius

Figure CN103620376BD00074

和R之间的范围内的环形光线中发出的光,则增量甚至可以达到70倍。 And an annular light rays within a range between the emitted R, then increment can even reach 70 times.

[0051] 半球状液滴是最优情况,但即使表面Sl偏离该标称形状,限制效应仍然存在。 [0051] semispherical liquid droplet is optimal case, but even if the surface Sl departing from the nominal shape, confinement effect persists. 为了量化可接受的偏离,首先必须考虑表面Sl可能并不精确地为球体的一部分,其次考虑通常为球形的物体G可能并不恰好在中间被截断或由完全平坦的表面截断。 To quantify the acceptable deviation, the first surface Sl may not be considered as a part of a sphere precisely, secondly consideration may often not exactly be truncated or cut off by a completely flat surface for the spherical object G in the middle.

[0052] 在图12A中可以看出,在S1的每个点P1,法线N1限定为朝向液滴的内部取向(从MA 至IJG)。 [0052] As can be seen in FIG. 12A, at each point P1 S1, the normal N1 is defined inside oriented towards droplets (from MA to IJG). 将相对于中心为C的球体的角度偏差定义为如下:Q1,err (Pi,C),其为Nl和直线(PI,C) 之间的角度。 With respect to the center C of the sphere is defined as the angle deviation: Q1, err (Pi, C), which is a straight line between Nl and (PI, C) of the angle. 最适于Sl的球面中心为点Cl,点Cl最小化 Sl most suitable spherical center point Cl, Cl minimized point

Figure CN103620376BD00075

(角度未被定向,因此其值总为正)。 (Non-oriented angle, so its value is always positive). 一旦已找到该点(被称为Sl的角中心),最大角度偏差Q^errMax被定义为在整个表面SI上的? Once this point has been found (to be referred to as center angle Sl), the maximum deviation angle is defined as Q ^ errMax over the entire surface of the SI? 1的01>(^ (Pi,c)的最大值。 01> (^ (Pi, c) a maximum value.

[0053] 将光限制在本发明所应用的液滴的边缘处的效应的程度取决于“光碗”的相对厚度,相对厚度为 [0053] The degree of light confinement effect at the edge in the present invention is applied depends on the relative thickness of the liquid droplets "optical bowl", the relative thickness of

Figure CN103620376BD00081

. 在球形表面的情况下: In the case of the spherical surface:

Figure CN103620376BD00082

[0054] 优选地,为了保持在光学几何区域,光碗的厚度必须大于光的波长λ。 [0054] Preferably, in order to maintain the optical geometry region, the bowl must be greater than the thickness of the light wavelength λ of the light. 换句话说,优选地, In other words, preferably,

Figure CN103620376BD00083

,对于波长λ为500nm,且假设next=l .33 (水的折射率),η=1 (空气的折射率),该条件导致 For a wavelength λ of 500 nm, and it is assumed next = l (refractive index of water) .33, η (refractive index of air) = 1, the condition causes

Figure CN103620376BD00084

. 此外,在通常情况下,液滴G的直径将大于2μπι,甚至大于4μπι。 Further, under normal circumstances, will be greater than the diameter of the droplets G 2μπι, even greater than 4μπι.

[0055] 如图12Β所示,在表面SI并不为上文所述的完全地球形的情况下,用于全反射的限制光线Rlim在角构成的最坏情况下,将与线段[P1,C1]成角度(0c+0errMax)。 [0055] FIG 12Β, in the case where the surface is not completely SI earth shaped as described above for total reflection of light to limit angle Rlim worst case consisting of the line segment [Pl, a C1] an angle (0c + 0errMax). 在该设置下,光碗的相对厚度为 In this arrangement, the thickness of the light relative to the bowl

Figure CN103620376BD00085

> 使用对应于空气中水滴的数值(IiintAie3xt= 4/3,即,0。=48.6°),将发现,0errMax=4.5°,对应于环宽度相对减小20 %。 > Value corresponding to the use of water droplets in the air (IiintAie3xt = 4/3, i.e., 0. = 48.6 °), will be found, 0errMax = 4.5 °, corresponding to a 20% reduction relative to the ring width. 如果能接受环宽度相对减小50%,则能够接受Sl相对于球体的角度偏差达12.5°。 If the width of the ring can accept relatively reduced by 50%, it is possible to accept the ball with respect to Sl angular deviation of 12.5 °.

[0056] 如图12C,标记为Θccint的(S1和S2之间的)接触角不是90 °意味着限制于光碗中的光束的主光线(RP)在通过S2时不再垂直于该表面。 [0056] FIG. 12C, labeled Θccint contact (between S1 and S2) meant to limit the angle is not 90 ° to the light beam of the principal ray of the bowl (RP) by S2 is no longer perpendicular to the surface. 定义公差的标准为,观察系统的整个数值孔径(图12D中阴影所示的峰值半角锥0ccmt)被包括在引导光束的孔径角中,该孔径角由RLl 和RL2限定。 Standard is defined tolerance, the numerical aperture of the entire system is observed (FIG. 12D shaded peak half angle cones 0ccmt) is included in the beam aperture angle of the guide, the angle defined by the aperture RLl and RL2. 仅考虑折射率为nint的介质,对于nint/rwt=4/3(水-空气对),引导光束的孔径角为ΘΝΑμμ= (9〇°-0c),g卩,为41.4°。 Nint considering only a refractive index of a medium, for nint / rwt = 4/3 (the water - air pair), direct the beam aperture angle ΘΝΑμμ = (9〇 ° -0c), g Jie, is 41.4 °. 对于包括在RLl和RL2之间的阴影三角形,则I 9〇°-0ccmt I〈 θNAMax-θ。 For shaded triangles comprised between RLl and RL2, then I 9〇 ° -0ccmt I <θNAMax-θ. bs是必要的。 bs is necessary. 对于具有典型的数值孔径NA=0.5的显微镜物镜所收集的光,在折射率为runt的介质中,孔径角0〇bs为22° (NA=nintsin9〇bs)。 For a typical numerical aperture NA = 0.5 microscope objective optical collected, runt a refractive index of the medium, the aperture angle 0〇bs of 22 ° (NA = nintsin9〇bs). 利用该可视化系统且对于mnt/next=4/3, 9cont在70°至110°的范围内。 And utilizing the visualization system for mnt / next = 4/3, 9cont in the range of 70 ° to 110 °.

[0057] 液滴G可以由大体圆形的固体或凝胶的凸体代替。 [0057] G may be replaced by a droplet substantially circular protuberance solid or gel. 在这些情况下,表面S2可以包括小平面(数目足够大,示例性值为至少10,优选为20或更大,以能够量化作为“大体圆形”的表面)。 In these cases, a small plane may include a surface S2 (a sufficiently large number, the exemplary value of at least 10, preferably 20 or more, as to be able to quantify "substantially circular" surface).

[0058] 周围介质并不必须是空气,但是空气这种选择是有利的,这是因为空气可以用来最大化mnt-next的差值。 [0058] surrounding medium need not be air but this choice is advantageously air, because the air can be used to maximize the difference mnt-next. 事实上,随着折射率之间的差值增大,优选地,runt^ I. IX next,光限制效应的效率增大。 In fact, as the difference between the refractive index increases, the efficiency Preferably, runt ^ I. IX next, light confining effect is increased.

[0059] 图6示出用于实施本发明方法的实验装置。 [0059] FIG. 6 shows the experimental apparatus for carrying out the method of the present invention.

[0060] 基板SS为170μπι厚的盖玻片,其被硅烷化以具有疏水性。 [0060] The thickness of the substrate SS is 170μπι coverslip, which is to have a hydrophobic silanated. 包含待检测对象或物质(微生物、在溶液中的荧光分子等)的水溶液被喷到该盖玻片上,形成半径为几μπι (例如2μπι 或4μπι)至200微米(S卩,体积约为IpL至IOnL)的液滴。 Or an aqueous solution containing a substance to be detected objects (microorganisms, fluorescent molecules, etc. in solution) is sprayed onto the cover glass, a radius of several μπι (e.g. 2μπι or 4μπι) to 200 micrometers (S Jie, to a volume of about IpL IOnL) droplets. 由于对表面进行了疏水化处理,这些液滴的接触角的范围为92°至96° (理想的半球状情况对应于90°的接触角)。 Because of the hydrophobic surface treatment, the contact angle of the droplets range of 92 ° to 96 ° (preferably hemispherical case corresponds to a contact angle of 90 °). 计算机模拟已经能够示出,重力不会使液滴变形,与盖玻片的取向无关。 Computer simulations have shown can be gravity does not deform the droplets, regardless of the orientation of the coverslip.

[0061] 应当注意,为了形成液滴,可以使用一些与简单的喷洒不同的技术,尤其是液滴要单分散时。 [0061] It should be noted that, in order to form liquid droplets, may be used with a number of different simple spraying techniques, in particular to monodisperse droplets. 可以提到的示例有:通过点样法沉积,即,通过使用微量吸移管(如,可以由自动装置控制的压电式的微量吸移管)沉积液滴(或多个液滴);通过分散法沉积;或如文献US6391578中所描述的通过微结构化亲水/疏水区进行沉积。 An example may be mentioned are: deposited by spotting, i.e., by using a micropipette (e.g., piezoelectric trace may be controlled by a robot pipette) depositing droplets (or droplets); by dispersing was deposited; or micro-structured by hydrophilic / hydrophobic regions described in document US6391578 deposited. 凝胶滴可以使用文献W082/ 02562中描述的技术进行沉积,其中,悬浮液在液体状态下被分馏,然后被凝胶化。 Gel droplets may use techniques described in the document W082 / 02562 is deposited, wherein the suspension is fractionated in a liquid state, it is then gelled.

[0062] 还可以设想使用一个或多个意欲填充液体样品或凝胶化样品的半球形杯。 [0062] It is also contemplated to use one or more of the liquid sample is intended to fill the sample gelled or hemispherical cup. 在这种情况下,杯的折射率小于其意欲包含的样品的折射率。 In this case, a refractive index less than the refractive index of the cup which is intended to contain a sample. 例如,在该配置中,样品可以是折射率通常大于1.4的离子液体。 For example, in this configuration, the sample may be generally greater than a refractive index of 1.4 ionic liquid. 因此,构成该杯的材料可以为折射率小于1.4且以基本上半球形的杯的中空块的形式的聚合物,或实际为薄的热成型膜。 Thus, the material constituting the cup can be less than 1.4, and in the form of a substantially hollow hemispherical cup block polymer refractive index, or actual thin thermoformable film. 在这两种情况下,杯子可以通过附接形成表面SS的透明盖而密封。 In both cases, the cup can be formed by attaching the transparent cover of the sealed surface SS.

[0063] 喷洒后,使盖玻片SS在借助双面胶带RDF形成的框架上翻转过来,该框架例如为“基因框架”类型(250μπι厚),且其已置于另一盖玻片L上。 [0063] After spraying the coverslips SS is turned over on the frame by means of double-sided tape RDF is formed, for example, the frame type "gene Framework" (250μπι thick), and which has been placed on the other coverslips L . 然后,胶带在盖玻片SS和盖玻片S 之间形成壁。 Then, the tape is formed between the wall and the cover glass coverslip SS S. 因此,形成薄的密封腔,其防止液滴蒸发。 Thus, a thin sealed chamber, which prevents evaporation of liquid droplets. 加热装置MC用来将该腔维持在恒定温度37°下,且组件被安装在机动化的台板PM上。 Means for heating the chamber MC is maintained at a constant temperature of 37 °, and the assembly is mounted on a motorized platen PM. 显微镜MS用来从上盖玻片SS这一侧观察液滴。 MS microscope cover glass for a droplet is observed from the side of the SS. 优选地,由样品所产生的光的照射和收集从相对于盖玻片SS的同一侧进行。 Preferably, the irradiation of light collected by the sample and generated from the same side with respect to the SS coverslip. 因此,样品的照射也可以受益于“光碗”中的限制效果,但这不是必须的。 Therefore, the illumination of the sample may also benefit from the effect of limiting the "light bowl", but this is not required.

[0064] 可以使用包括物镜LO (例如:放大倍数10,数值孔径0.5)和用于图像采集的照相机CM的成像系统进行观察。 [0064] The objective lens may be used include the LO (e.g.: 10 magnification, numerical aperture 0.5) and the imaging system used for image acquisition camera CM was observed. 在图6中,附图标记SO通常表示成像系统的其它元件(照明装置、分色镜、滤波器等),其为可变的并取决于所采用的观察技术。 In FIG. 6, the reference numeral generally represents SO other elements of the imaging system (illumination means, dichroic mirrors, filters, etc.), observation technique which is variable and depends employed. 事实上,本发明的原理可以应用到一些不同的观察技术,将在下文结合图7A至图12更详细地描述这一点。 Indeed, the principles of the present invention may be applied to a number of different observation technique, in conjunction with FIGS. 7A to FIG. 12 described below in more detail it. 显然地,该实施方式列表并不详尽。 Obviously, this list is not exhaustive embodiment. 为简单起见,图7A至图12未示出光学系统或用于实施该方法所使用的图像学。 For simplicity, FIGS. 7A to FIG. 12, not shown optical system for carrying out the image learning method used.

[0065] 图7A以高度图解的方式示出本发明的方法应用至通过落射荧光显微技术检测液滴中的荧光团。 [0065] FIG 7A shows a highly diagrammatic manner a method of the present invention is applied to a group by epifluorescence microscopy to detect fluorescence in droplets. 波长为1:的激发光由光源SL (通常是配备有滤波器的汞灯或卤素灯)发出, 经由液滴G的附着到基板SS的表面S2被射入液滴G中。 Wavelength 1: excitation light from a light source SL (usually mercury lamp equipped with a filter or halogen) emitted is incident on the droplet in a droplet G G attached to the surface of the substrate S2 via the SS. 部分被限制在“光碗” BL中的该光诱导焚光团的焚光性,焚光团发出波长为λ2>λι的福射。 Burning light moiety is limited "optical bowl" BL in the light of the light-induced group-burning, the burning light group emitted wavelength λ2> λι exit blessing. 从图7B中可以看出,焚光福射离开液滴和基板,部分地聚集在发光环中。 As it can be seen from FIG. 7B, burning away the light emitting fu droplets and the substrate, partially accumulated in the illuminated ring. 应当注意,光碗外的荧光团分子对照相机CM检测到的信号起到的效用较小,这是由于其暴露在强度较低的激发光的通量中,并且其发射的光子没有被限制在发光环内。 It should be noted that small molecule fluorophores for the outer bowl light camera CM detection signal utility functions, this is due to the exposure to the flux of the lower intensity of the excitation light, and emits a photon is not limited hair in the ring. “光碗”所占据液滴体积的部分随着比的幅度的增大而增大。 Droplet volume "light bowl" occupied portion with increasing amplitude ratio increases.

[0066] 图7B的图像是通过WA1=SeOnm的波长激发荧光团4MU在MOPS缓冲液(3-(N-吗啉代)丙磺酸)中的溶液而获得的。 Image [0066] FIG. 7B is WA1 = SeOnm by the wavelength of the excitation fluorophore 4MU in MOPS buffer (3- (N- morpholino) propanesulfonic acid) solution obtained. 通过以λ2=450ηπι为中心的滤波器分离荧光辐射。 By separating fluorescence radiation λ2 = 450ηπι centered filter.

[0067] 在变型中,可以经由表面Sl照射液滴,这导致液滴在空间上更均匀的激发,从而损害将激发束限定在光碗中的效应。 [0067] In a variant, the droplet can be illuminated via surface Sl, which results in a more spatially uniform droplet excitation, thereby impairing the excitation light beam is defined bowl effect. 在这种情况下,不再可以使用术语“落射荧光”。 In this case, no longer use the term "epi-fluorescence."

[0068] 图7Α和图7Β的观察方法尤其可以应用于化学反应或生化反应的动力学研究。 Observation method [0068] FIGS 7Α and 7Β particular kinetics of the chemical reaction or biochemical reaction may be applied.

[0069] 图8Α和图8Β涉及仍然使用落射荧光显微术原理对微观尺寸的荧光对象E (如细菌, 在适当的情况下,转基因的细菌)进行观察的情况。 [0069] and FIGS. 8Α 8Β relate still using epifluorescence microscopy principle of fluorescence microscopic size of the object E (such as bacteria, in appropriate cases, bacterial gene transfer) was observed situation. 如果对象E的密度比形成液滴的液体的密度大,其将朝着液滴的底端下落,随后将其自身定位在“光碗”内。 If the density ratio of the density of the object E is formed large droplets of liquid, which droplets falling towards the bottom end, then position itself in the "light Bowl." 因此,将激发光限制在表面Sl附近被证实是特别有利的。 Thus, the excitation light confinement has proven to be particularly advantageous in the vicinity of the surface Sl. 图8Β示出其中对象E发出的光辐射通过液滴/周围介质的界面而聚集并且引导的环以及由之前不被表面Sl反射而是穿过基板的“直接”辐射所形成的所述对象的图像。 And said object ring "directly" by the radiation through the substrate, but not before the reflecting surface Sl shown in FIG 8Β wherein the object light radiation E emitted by a droplet gathered interface / media surrounding and guiding the formed image.

[0070] 在该图8Β的情况下,对象E是包括MUG (4MU葡萄糖醛酸酯)的生长培养基中的转基因的大肠埃希氏杆菌细菌。 [0070] In the case of this FIG 8Β, the object E is Escherichia coli bacteria growth medium comprising a transgene MUG (4MU glucuronic acid ester) in. 在Xi=557nm (激发波长)和λ2=579ηηι (焚光波长)下进行观察。 In the observation Xi = 557nm (excitation wavelength) and at λ2 = 579ηηι (burning light wavelength).

[0071] 根据图8Α和图8Β的观察方法可应用于对接种到溶液中的细菌进行计数,该溶液如上述喷洒以在显微镜用载玻片上形成一系列液滴,细菌以随机方式分布在液滴中。 [0071] The vaccination can be applied to the bacterial solution is counted according to the observation method of FIG 8Α and 8Β of the solution was sprayed as described above to form a series of droplets on the microscope slide, the bacteria were distributed in a random manner drop in. 该溶液可以包括参考荧光团,例如FITC(异硫氰酸荧光素),其意味可以对液滴进行计数、估算它们的体积,以及对来自细菌的荧光信号进行归一化以消除仪器漂移。 The solution may include a reference fluorophore, e.g. FITC (fluorescein isothiocyanate), which means that the droplet can be counted to estimate their size, and the fluorescent signal from the bacteria is normalized to eliminate instrument drift.

[0072] 使用大肠埃希氏杆菌ATCCl 1775 (其为β-葡萄糖苷酸酶阳性的菌株)的菌株测试该方法。 [0072] Escherichia coli using ATCCl 1775 (which is a positive strains β- glucuronidase) test method strain. 葡萄糖苷酸酶的酶活性可用于降解荧光基质MUG,这导致荧光团4MU的释放。 Glucuronidase activity may be used for the degradation of a fluorescent substrate MUG, which results in the release of 4MU fluorophore. 接种有这些细菌的水溶液为包含MOPS (3- (Ν-吗啉代)丙磺酸)、荧光基质MUG和作为参考荧光团的FITC的缓冲液。 These bacteria inoculated with an aqueous solution containing MOPS (3- (Ν- morpholino) propanesulfonic acid), and the reference fluorescent substrate MUG fluorophores FITC buffer. 该介质的组成如下: The composition of the medium is the following:

[0073] -150mM的MOPS (4-吗啉代丙磺酸,钠盐); [0073] -150mM of MOPS (4- morpholino propanesulfonic acid, sodium salt);

[0074] -2.6mM 的硫酸镁; [0074] -2.6mM magnesium sulfate;

[0075] -854μΜ的葡萄糖醛酸钠; [0075] -854μΜ of sodium glucuronate;

[0076] _870μΜ的甲基β-d-葡糖苷酸; [0076] _870μΜ methyl β-d- glucuronide;

[0077] -113μΜ的MUG (4-甲基伞形酮-β-d-葡糖苷酸); [0077] -113μΜ of MUG (4- methylumbelliferyl glucuronide -β-d-);

[0078] -4·15μΜ 的荧光素。 [0078] -4 · 15μΜ fluorescein.

[0079] 使用具有物镜(针对载片厚度对其进行校正,χ10,ΝΑ0.5)的Zeiss的Axiolmager显微镜对液滴进行观察。 [0079] using an objective lens (for a thickness of the carrier sheet be corrected, χ10, ΝΑ0.5) of a Zeiss microscope Axiolmager droplets observed. MUG作为每个细菌的标记,这是由于包括至少一个细菌的液滴中产生4MU荧光团。 As each marker MUG bacteria, since bacteria comprises at least one droplet generating 4MU fluorophore. 荧光团FITC作为每个液滴的标记,这是由于每个液滴发出FITC的光,而不取决于细菌的存在。 Fluorophores FITC labeled as each droplet, because each droplet FITC emits light without depending on the presence of bacteria.

[0080] 由于荧光团4MU由细菌的新陈代谢产生,故其荧光信号随时间而增强。 [0080] Since 4MU fluorophore produced by the metabolism of the bacteria, so the fluorescent signal increases with time. 此外,该缓冲液包括给定浓度的荧光团FITC,其在整个测量中保持不变:因此,来自FITC的荧光信号为恒定的,且可以用作参考信号。 In addition, the buffers include fluorophores FITC given concentration, which remains constant throughout the measurement: Thus, the fluorescence from the FITC signal is constant, and can be used as a reference signal.

[0081] 在观察期间: [0081] In the observation period:

[0082] -交替地以FITC荧光团激发波长和4MU荧光团激发波长激发样品(待完成); [0082] - alternately FITC fluorophore excitation wavelength of the fluorophore and the excitation wavelength of the excitation 4MU sample (pending);

[0083] -交替地记录对应于FITC的发射波长的图像和对应于荧光团4MU的发射波长的图像; [0083] - alternately recorded corresponding to the emission wavelength of FITC emission image corresponding to a wavelength of the fluorophore 4MU image;

[0084] -在每个FITC发射图像上定位全部发荧光的液滴,每个液滴在图像上产生环。 [0084] - positioning the entire fluorescent FITC droplets on each emission image, each drop is generated on the image ring. 每个环限定对应于每个液滴的所关注的空间区域。 Each ring defines a space corresponding to the region of interest of each droplet. 另一方面,可以测量给定图像的每个环的外半径Rext,使用以下表达式表示该半径与每个液滴的体积的关系: On the other hand, it can be measured to an outer radius of each ring Rext given image, using the following expression showing the relationship of the volume of each droplet radius:

[0085] [0085]

Figure CN103620376BD00101

[0086] 其中: [0086] wherein:

[0087] -0_t=液滴在盖玻片SS上的接触角; [0087] -0_t = contact angle of the droplet on coverslips SS;

[0088] -Rext=发光环的外直径。 [0088] -Rext = outer diameter of the light ring.

[0089] 所关注的预限定的空间区域用在每个4MU荧光团发射图像上。 [0089] The pre-defined spatial region of interest on each of the used fluorophores 4MU emission image. 确定来自构成每个所关注的空间区域的像素的信号的平均强度,该平均强度为每个液滴产生的信号的平均强度。 Determining an average intensity of the signals from the pixels constituting each spatial region of interest, the average intensity of the average intensity of the signal generated by each drop. 可选地,在荧光团4MU的发射波长下每个液滴所产生的信号的平均强度可以通过在FITC 的发射波长下对应于同一液滴的信号的平均强度归一化。 Alternatively, an average intensity signal at the emission wavelength of the fluorophore 4MU each droplet can be generated by a signal corresponding to the same droplet at the emission wavelength of FITC average intensity normalized. 通过对强度或归一化强度定阈值,确定包括至少一个细菌的液滴,定阈值允许关于液滴中存在或不存在至少一个细菌做决定。 Or by the intensity of normalized intensity given threshold, determining at least one bacterium comprising droplets of liquid droplets on a predetermined threshold value to allow the presence or absence of at least one bacterium to make a decision.

[0090] 通过应用泊松定律,可以确定最初溶液(S卩,以液滴形式分散的溶液)中的最可能的细菌浓度(CPP)。 [0090] By applying the Poisson Law, most likely bacterial concentration (CPP) may determine that the original solution (S Jie, in the form of droplets dispersed solution).

[0091] 可以证明: [0091] can be proved:

[0092] [0092]

Figure CN103620376BD00111

[0093] -Vj为每个被至少一个细菌占据的液滴j的单元体积; [0093] -Vj cell volume j for each droplet being occupied by at least one bacteria;

[0094] -Vi为通过FITC荧光观察的每个液滴i的单元体积; [0094] -Vi unit volume of each droplet is observed by FITC fluorescence of i;

[0095] -CPP为初始溶液中最可能的细菌浓度。 [0095] -CPP most likely solution as the initial concentration of bacteria. 该浓度的大小为体积的反演计算。 The size is calculated as the volume concentration of inversion. 在试验中,在不到4小时的时间里,确定值为5.7 X IO5个细菌/mL。 In the test, in less than four hours, the determined value of 5.7 X IO5 bacteria / mL. 确切的细菌浓度未知,因此,在大于24小时的培养后,实施CPS3盘计数(供应商:bioM6rieux)作为参考。 The exact concentration of bacteria is unknown, and therefore, at greater than 24 hours of incubation, the plate counts embodiment CPS3 (Supplier: bioM6rieux) as a reference. 该计数方法提供的结果为6.5X IO5个细菌/mL。 The result of this counting method for providing 6.5X IO5 bacteria / mL. 该结果与参考方法有良好的一致性,这是由于观察差异实际上小于2倍,这是通常可以接受的。 This result is in good agreement with a reference method, which is due to the observed differences in fact less than 2 times, which is usually acceptable.

[0096] 图9A和图9B示出通过反射显微镜检查法(以相同的波长λ穿过基板SS而进行照射和检测)观察到的液滴的图像。 Image droplets [0096] FIG. 9A 9B shows (illumination and detection is performed at the same wavelength λ through the substrate SS) by a reflection microscopy and observed. 例如,这可以用于验证微流体器件的功能,研究基板的润湿性,或研究形成液滴的液体的折射率。 For example, this can be used to verify the function of a microfluidic device, Wettability of the substrate, the refractive index of the liquid or research droplets.

[0097] 在观察生物学对象或分析物时,可以利用在液滴与周围介质之间的界面处所发生的光导引,以使用光学光谱方法(例如,固有荧光和非弹性的拉曼散射)提供增强的灵敏性。 [0097] When viewed object or biological analytes may be guided by the light spaces between the interface with the surrounding medium the droplets occurs, using optical spectroscopic methods (e.g., intrinsic fluorescence and inelastic Raman scattering) provide enhanced sensitivity. 对于分析物,使用与荧光相同的配置(图7Α)。 For analytes using fluorescent same configuration (FIG 7Α). 对于单个对象Ε,可以使用荧光配置(图8Α)以及其中激发光限于入射到对象上的笔形光束的一种新的配置(参见图10)。 For single object Ε, as a fluorescent configuration (FIG. 8a) and wherein the excitation light is limited to a pen-shaped light beam is incident on the object a new configuration (see FIG. 10).

[0098] 目前所描述的所有观察方法涉及通过凸表面Sl或通常为优选地通过平坦表面S2 照射样品。 [0098] All current observation method described relates generally convex surface Sl or flat surface, preferably by irradiating the sample S2. 然而,当样品发光时,这种类型的照射是不必要的,一些微生物或其中发生使得发光的化学反应(由诸如碱性磷酸酶或辣根过氧化物酶的酶催化的化学发光反应)的一些溶液或包括荧光素酶活性的改性的微生物(例如,在ML Eldridge等所著文章aSaccharomyces cerevisiae BLYAS,a New Bioluminescent Bioreporter for Detection of Androgenic Compounds”,APPLIED AND ENVIRONMENTAL MICR0BI0L0GY73 (19),第6012-6018页(2007)中所述的)就是这种情况。进行模拟以在以下两种配置中比较置于离液滴有一定距离的检测器以均匀且各向同性的方式收集液滴内部产生的光信号而检测到的光信号: However, when the sample emission, this type of irradiation is not necessary, so that the occurrence of some microbes, or wherein the light-emitting chemical reaction (chemiluminescence by the reaction of the enzyme such as alkaline phosphatase or horseradish peroxidase enzyme-catalyzed) of some of the solution comprising a microorganism or modified luciferase activity (e.g., in the article written by ML Eldridge et aSaccharomyces cerevisiae BLYAS, a New bioluminescent Bioreporter for Detection of androgenic Compounds ", APPLIED AND ENVIRONMENTAL MICR0BI0L0GY73 (19), the first 6012-6018 light page (2007) above) is the case. simulation to compare the droplet is placed at some distance from the detector to collect two configurations droplet generator inside a uniform and isotropic manner signal, the optical signal detected:

[0099] -如图3A所示的半球状液滴,该液滴由包括给定浓度的化学发光分子的水 [0099] - a hemispherical droplet as shown in FIG. 3A, the liquid droplet including a given concentration of water chemiluminescent molecules

[0100] 溶液形成; [0100] forming a solution;

[0101] -图4A所示的配置中的浸在油浴中的具有相同体积且相同组成的球状液滴。 [0101] - the configuration shown in FIG. 4A is immersed in an oil bath having the same volume and the same composition of the spherical drops.

[0102] 根据计算,对于数值孔径在0.1到0.6范围内的检测器,当液滴为半球状时,所收集的信号高出2.5倍至2.8倍。 [0102] The calculations for the numerical aperture of the detector in the range of 0.1 to 0.6, when a droplet is hemispherical, the collected signals 2.5 to 2.8 times higher. 因此,半球状配置产生更强的信号。 Accordingly, hemispherical configured to generate a stronger signal.

[0103] 图11涉及特定的实例,其中,在其表面Sl上沉积有官能化层CF的半球状(“半珠”) 的固体突起V被用作化学传感器。 [0103] FIG. 11 relates to a specific example in which, on the surface of the functional layer is deposited Sl CF hemispherical ( "hemi-beads") was used as a solid projection V a chemical sensor. 形成或包括在周围介质MA(液态或气态)中的化学物质与官能化层CF反应以产生可检测的光信号(如荧光信号或发光信号)或改变官能化层CF的折射率,这进而影响发光环的内半径(在图9A的设置中的观察)。 Include chemical substances or are formed with the functional layer in the surrounding medium CF MA (liquid or gaseous) in reaction to an optical signal (such as a fluorescent signal or emission signal) generated detectable or changing the refractive index of the functional layer CF, which in turn impact hair inner radius halo (viewed in the arrangement of FIG. 9A). 在变型中,构成突起V的材料本身可能与周围介质反应。 In a variant, the material constituting the projections V themselves be the reaction with the surrounding medium. 在此情况下,突起可以由液滴替换或由凝胶构成。 In this case, the protrusion may be replaced by or constituted by a gel droplet.

Claims (9)

  1. 1. 一种用于观察样品的方法,所述方法包括如下步骤: a) 提供基本上透明的介质形成的物体(G,V),所述介质具有第一折射率(mnt)且具有圆形或多平面的第一表面(SI)和基本上平坦的第二表面(S2),所述第一表面将所述物体与周围介质(M)分离开,所述周围介质具有低于所述第一折射率的第二折射率(nMt),待观察的所述样品位于所述物体内或沉积在所述第一表面上; b) 通过所述基本上平坦的第二表面观察所述样品,所述步骤b)包括用于照射所述物体的操作,并且所述步骤b)包括检测荧光信号或者拉曼散射信号或者所述步骤b)利用反射显微术或透射显微术进行;以及c) 通过在所述步骤b)期间检测与所述物体的光碗相匹配的发光环,确定所述样品中的物质或对象的存在, 所述物体是液滴且所述物体通过在所述光碗(BL)内由所述样品沿着所述第一表面散射的光被照 1. A method for observing a sample, said method comprising the steps of: a) providing a substantially transparent article (G, V) formed in the medium, said medium having a first refractive index (mnt) and having a circular a first surface or plane (SI) and a substantially planar second surface (S2), the first surface of the object and the surrounding medium (M) separated from the surrounding medium having a lower than the first a refractive index of the second refractive index (NMT), the sample to be observed or the object is located within deposited on the first surface; b) the sample through said second substantially planar surface observation, said step b) comprises an operation for illuminating the object, and said step b) comprises detecting the fluorescent signal or Raman scattering signal or the step b) using the reflection or transmission microscopy for microscopy; and c. ) by detecting the light bowl object during said step b) to match the illuminated ring, determining the presence of a substance in a sample or subject, and the object is a droplet by the light of the object the bowl (BL) along the first surface scattered by the sample light is illuminated 射,用于照射所述物体的所述光从所述第二表面经由所述发光环穿入所述液滴中,并且其特征在于,所述光碗的相对厚度限定为: -如果所述第一表面是球形的表面,则所述相对厚度等于 Emitting means for irradiating the object light from the second surface of the light rings via the penetrating droplet and wherein the relative thicknesses of the light bowl defined as: - if the the first surface is a spherical surface, then the relative thickness equal to
    Figure CN103620376BC00031
    5和-如果所述第一表面不是完全球形的表面,则所述相对厚度等于 5 and - if the first surface is not entirely a spherical surface, then the relative thickness equal to
    Figure CN103620376BC00032
    其中,Θ。 Which, Θ. 表示光线的入射临界角,表示最大角度偏差。 Represents a critical angle of incidence of light, it represents the maximum angular deviation.
  2. 2. 根据权利要求1所述的方法,其中,所述物体具有基本上半球形的形状。 The method according to claim 1, wherein said object has a substantially hemispherical shape.
  3. 3. 根据权利要求1或2所述的方法,其中,所述步骤c)包括确定所述样品中的微生物的存在。 3. The method of claim 1 or claim 2, wherein said step c) comprises determining the presence of microorganisms in the sample.
  4. 4. 根据权利要求1或2所述的方法,其中,所述液滴附着到透明基板(SS)上而形成所述基本上平坦的第二表面。 4. The method of claim 1 or claim 2, wherein the droplets adhere to a transparent substrate (SS) is formed of the second surface is substantially planar.
  5. 5. 根据权利要求4所述的方法,其中: -所述步骤a)包括产生形成多个所述物体的多个液滴,所述多个液滴中的至少一些液滴包括至少一个待检测的对象或物质;以及-所述步骤b)包括同时观察多个所述液滴。 5. The method of claim 4, wherein: - said step a) comprises generating a plurality of droplets of said plurality of objects, at least some of the plurality of droplets comprise at least one droplet to be detected object or substance; and - said step b) comprises a plurality of said droplets were observed.
  6. 6. 根据权利要求1或2所述的方法,其中,所述物体由对分析物敏感的材料形成,所述分析物包括在所述周围介质中。 The method according to claim 1 or claim 2, wherein the object is formed from a material sensitive to the analyte, the analyte included in the surrounding medium.
  7. 7. 根据权利要求1或2所述的方法,其中,所述周围介质是空气。 7. The method of claim 1 or claim 2, wherein said surrounding medium is air.
  8. 8. 根据权利要求1所述的方法,其中,所述照射从所述基本上平坦的第二表面这一侧进行。 8. The method according to claim 1, wherein the illumination from said second substantially planar surface for this side.
  9. 9. 根据权利要求1所述的方法,其中,所述步骤b)通过落射荧光显微术进行。 9. The method according to claim 1, wherein said step b) is performed by epifluorescence microscopy.
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